07-10【High-Efficient Ligation Reagent】

Ligation high Ver.2(Code No. LGK-201)

Instruction Manual

TOYOBO CO., LTD. Life Science Department
OSAKA JAPANA3706K

0INDEX[１] Introduction........................................................................................ 1[２] Contents ............................................................................................ 2[３] Protocol ............................................................................................. 3[４] Application data
(1) Cohesive end ligation.............................................................. 4(2) Blunt end ligation..................................................................... 4(3) Linker ligation .......................................................................... 5(4) TA cloning using purified PCR products.................................. 5(5) TA cloning using unpurified PCR products.............................. 6(6) Phage vector ligation............................................................... 6[５] Trouble shooting ................................................................................ 7●Caution
This product is for research use only. Never use this product for diagnostic purposes or forother clinical purposes. The potential hazards that may be caused by any component of thisproduct have not been fully investigated. Adequate care should be taken when handling thisproduct, e.g., use protective equipment and clothing.

1[１] Introduction
The ligation reaction is an essential step in genetic recombination experiments.
For this reaction, T4 DNA ligase has been widely used. Ligation high Ver.2 is ahighly efficient premixed T4 Ligase reagent.
Features of Ligation high Ver.2

12010080Blue
White

60
4020Ve
high
Liga
tion

Com
petit
or
ACom
petit
or
B

0r.2

Relative colony number● Highly efficient*
Effective ligation of cohesive, blunt and A overhang DNA fragments can beachieved.

Fig. 1 Comparison of TA cloning efficiency
Colony number of Ligation high Ver.2 corresponds to 100● Will not freeze at -20℃*
Just use. No need to thaw.
● Ready to use
Just mix ligation high Ver.2 with an equal volume or with double the volumeof the solution containing DNA fragments.
*Patent pending1[２] Contents●Contents
750 μl × 1Vial (100 rxn)

Ligation high Ver.2●Storage conditions-20℃

R elative colony num ber※In the case of -30℃ storage or long-term storage under -20℃, the reagentmight freeze. No decrease in reaction efficiency is observed following 5freeze-thaw cycles.

12010080

B lue
W hite6040
20005Times of Freezing and thaw

Fig. 2 Effect of freezing and thaw
for activity of Ligation high Ver.2
Ligation efficiency was estimated by the TA cloning test.
Colony number at 0 times of freezing and thaw corresponds to 100
No negative effect was not detected by 5-time-freezing and thaw.

2[３] Protocol
Vector DNA＋Insert DNA*1
Ligation high Ver.2
16℃、30 min.*3*4

7.5 μl3.75～7.5 μl*2↓Transform 100 μl competent cells using ～10 μl reaction solution *5*1 Ligation efficiency is decreased by adding excess salts. DNA fragments should be in low saltsolutions (e.g. 10 mM Tris-HCl[pH 8.0], 1 mM EDTA)*2 Adding an equal volume of Ligation high Ver.2 to the DNA solution is recommended in the case ofTA cloning.
*3 The reaction can be prolonged for up to 2 hr. Standard cohesive end ligations can be completedwithin 5 min.
*4 The reaction can be performed at 4℃～25℃.
*5 The volume of ligation reaction added should be less than 10% of the volume of the competent cellsuspension. For electro-transformation, ligation reactions should be purified, because salts inhibit
the transformation.

Table 1 Recommended reaction conditions
DNA ： Ligation reagent
Reaction condition(Volume)Cohesive end ligation
1 ： 1 or 1 ： 0.5
16℃、5～30 min
Blunt end ligation
1 ： 1 or 1 ： 0.5
16℃、30 min
Linker ligation
1 ： 1 or 1 ： 0.5
16℃、30 min
T vector ligation
1 ： 116℃、30 min（Purified PCR procuct）
T vector ligation *1*2
1 ： 116℃、30 min（Unpurified PCR product）
Phage vector ligation
1 ： 1 or 1 ： 0.5
16℃、30 min*1 When PCR reactions contain primer dimers or unexpected PCR products, the desired
products should be purified.
*2 When sufficient amplification is confirmed, 0.5～1 μl of PCR product can be useddirectly. The reaction conditions are as follows;
H20
PCR product
Ligation high Ver.2

X
0.5～1
7.5
15 (μl)3[４] Application data
(1) Cohesive end ligation

Relative colony number

Dephosphorylated pUC19/HindIII,（50 ng, 25 fmol,）was mixed, at different ratios, withthe 546 bp DNA fragment of HindIII digested phage lambda DNA. An equal volume ofLigation high Ver.2, 7.5 μl, was added and incubated at 16℃ for 30 min. E. coli DH5
α competent cells were transformed using 10 μｌ of reaction mixture, and cultured
on LB/Amp(X-Gal, IPTG) plates O/N.

120
100
80
60
40
20
0

1：
5

1：
3

1：
1

1：
0.5

Blue
WhiteVector ： Insert （molar ratio）
Fig. 3 Cohesive end ligation
Colony number at 1:5 corresponds to 100(2) Blunt end ligation
Dephosphorylated pUC19/HincII,（50 ng, 25 fmol,） was mixed, at different ratios, withpurified PCR product (500 bp: from high-fidelity PCR enzyme*1). Half or an equalvolume of Ligation high Ver.2, 3.75 or 7.5 μl, was added and incubated at 16℃ for30 min. E. coli DH5α competent cells were transformed using 10 μｌ of reaction
mixture, and cultured on LB/Amp(X-Gal, IPTG) plates O/N.
*1

1：
5

1：
3

1：
1

.5

200Blue
White

20
0

1：
540

80
60
40

1：
360

100

1：
180

120

.5

100

1：
0

Relative colony number

120

1：
0

Relative colony numberHigh-fidelity PCR enzyme generates blunt ends.

Vector ： Insert （molar ratio）

Vector ： Insert （molar ratio）

DNA ： Ligation high Ver.2 = 1：1

DNA ： Ligation high Ver.2 = 1：0.5

Fig. 4 Blunt end ligation
Colony number on the condition of DNA ： Ligation high Ver.2 = 1：0.5 and Vector ：Insert = 1：3
corresponds to 100.

4

(3) Linker ligation

Relative colony number

120
100
80
60
40
20

120
10080
Blue
White

60
40
20

1：
10

1：
5.5
1：
01：
10

1：
5

1：
31：
1.5
1：
0Vector ： Insert （molar ratio）

1：
3001：
1

Relative colony number

Dephosphorylated pUC19/HincII,（50 ng, 25 fmol, was mixed at different ratios with ）phosphorylated HindIII linker (10mer). Half or an equal volume of Ligation high Ver.2,
3.75 or 7.5 μl, was added and incubated at 16℃ for 30 min. E. coli DH5αcompetent cells were transformed using 10 μｌ of reaction mixture, and cultured on
LB/Amp(X-Gal, IPTG) plates O/N.

Vector ： Insert （molar ratio）

DNA ： Ligation high Ver.2 = 1：1

DNA ： Ligation high Ver.2 = 1：0.5

Fig. 5 Linker ligation
Colony number on the condition of DNA ： Ligation high Ver.2 = 1：0.5 and Vector ：Insert = 1：10
corresponds to 100.

(4) TA cloning using purified PCR prodcuts

R elative colony num ber

T vector,（50 ng, 25 fmol,） was mixed, at different ratios, with purified PCR products(500 bp from Taq DNA polymerase). An equal volume of Ligation high Ver.2, 7.5 μl,
was added and incubated at 16℃ for 30min. E. coli DH5α competent cells weretransformed using 10 μｌ of reaction mixture, and cultured on LB/Amp(X-Gal, IPTG)
plates O/N.
120
100806040200B lue
W hite

1：0.5

1：1

1：3

1：5

Vector ：Insert （molar ratio）

Fig. 6 TA cloning using purified products
Colony number at 1:5 corresponds to 1005(5) TA Cloning using unpurified PCR products

Relative colony number

T vector,（50 ng, 25 fmol,） was mixed, at different ratios, with unpurified PCR products(500 bp from Taq DNA polymerase). An equal volume of Ligation high Ver.2, 7.5 μl,
was added and incubated at 16℃ for 30 min. E. coli DH5α competent cells weretransformed using 10 μｌ of reaction mixture, and cultured on LB/Amp(X-Gal, IPTG)
plates O/N.

120
100
8060
40
200BlueWhite210.5

Amount of the added PCR products（μl）

Fig. 7 TA cloning using unpurified products
Colony number on the condition of adding 0.5μlof the PCR product corresponds to 100.

(6) Phage vector ligation
λZAP®II (500 ng), EcoRI digested, and pRheo/EcoRI test insert（225 ng, 2.8 kb）,
were mixed with half or with an equal volume of Ligation high Ver.2, 3.75 or 7.5 μl,and incubated at 16℃ for 30 min. in vitro packaging was performed with 1.5 μlligation reaction using GIGAPACK®III（Stratagene,） followed by infection of E. coli
XL-1 Blue MRF’. The E. coli cells were cultured O/N and plaques counted.
Table 2. Reaction conditions and efficiency
DNA soln：
1 ： 0.5
1 ： 1
Ligation reagent
Efficiency
6.3×106
5.5×106(pfu/μg ZAP®II)

6

[５] Trouble shooting
TroubleNo or lowcolony numberCause
Excess amount ofligation mixture fortransformation
Vectorconcentration is low

Counter measures
Unpurified ligation mixtures should be addedat less than 10%(V/V) of competent cellsuspensions.
Increase the vector concentration.
(→See p4～6)

DNA insert
fragment
concentration is toolow.
Excess salts

Increase the DNA insert fragment
concentration.
(→See p4～6)

Ends of both vector
and insert aredephosphorylated.
Cloning
efficiency waslow

Primer dimer

・DNA fragments should be disolved in alow salt solution.
・ Unpurified PCR products should beadded at 0.5～1 μl per ligation reaction.
Dephosphorylated vectors cannot ligate withunphosphorylated PCR products.
When the PCR reaction contains primer
dimers or unexpected PCR products, thedesired PCR product should be purified.

Insufficient time for The reaction can be prolonged for up to 2hr.
ligation reaction.
Insertfragment Increase the insert fragment concentration.
concentration is too (→See p4～6)
low.
Self-ligation ofvector

Use dephosphorylated vector.

Excess number Self-ligation ofof colonies
vectorUse dephosphorylated vector.

Freezing of the Storage conditions
reagent

In the case of -30℃ storage or long-term
storage under -20℃, the reagent mightfreeze. No decrease in reaction
efficiency is observed following 5
freeze-thaw cycles. .

7

TOYOBO CO., LTD.
Life Science Department
2-8, Dojima Hama 2-chome, Kita-ku, Osaka 530-8230, Japan8